In order to examine the problems presented by the background which follows, one inventor inaugurated a study as yet unpublished called: “The effect of liposomal glutathione on the oxidation of the cholesterol components known as Low density lipoprotein (LDL) and high density lipoprotein (HDL).” Observations with respect to the interaction between glutathione peroxidase, liposomal reduced glutathione, and HDL and LDL led to this invention. In particular, the study highlighted the importance of reduced glutathione in maintaining the normal state of function of HDL and LDL, apparently by lowering the oxidation state of LDL and HDL. Other antioxidants may provide indirect support for normal function, but reduced glutathione functions much more efficiently and effectively. Further, it was determined that synergistic effect could be achieved by potentially using lower doses of statins than are usually required, reducing side effects while sustaining the intended function of the statin. Moreover, the addition of CoQ10 or ubiquinone could ameliorate one of the side effects of statins.
It was estimated in 2001 that 60 million Americans had heart disease (Lefkowitz). This number translates to a statistic that more than 1 out of every 5 individuals has heart disease. While an array of interventions, both medical and surgical have reduced the death rates in the decade ending 1997 by 20%, 12.2 million Americans have a history of heart attack, chest pain or both. With 1 million in the US dying of cardiovascular disease each year, it remains that nation's leading cause of death.
Concepts of related to the development of atherosclerosis are evolving. Elevations of the fatty material in blood such as cholesterol have been associated with increased risk of coronary artery disease (Castelli). However, more than half of the individuals with coronary artery disease have total levels of cholesterol that are less than the level considered normal, that is <200 mg/dL (Lavie). Thus, additions to the current methods of management of atherosclerosis are needed to change the prevalence of this disease.
Three theories of the cause of atherosclerosis have emerged recently in regard to the cause of atherosclerosis. Their emphasis is overlapping in terms of the events necessary to support the development of atherosclerosis (Stocker) and is reviewed in detail in Stocker's article (Stocker R, Keaney J F Jr. Role of Oxidative Modifications in Atherosclerosis. Physiological Review, Oct. 1, 2004; 84(4): 1381-1478. PMID: 15383655 http://physrev.physiology.org/cgi/content/full/84/4/1381).
These theories in include:
                Response to injury: This refers to damage to the arterial lining cells called endothelial lining cells by various factors such as shear stress, infection and inflammation, and even the mechanical trauma of interventions such as arterial stent placement.        Response to retention: This refers to the concept that fatty materials such as lipoprotein retention is the inciting event for atherosclerosis. The emphasis is on the retention of a particular lipoprotein called Low Density Lipoprotein (LDL).        Oxidative modification: This hypothesis focuses on the concept that LDL in its native, that is non-oxidized state, is not atherogenic. However, LDL modified by chemical reactions results in the oxidized form of LDL or oxidized LDL (ox LDL). OxLDL is readily internalized by macrophages and can lead to the formation of foam cells and the sequence of events associated with the formation of atherosclerosis.        
Oxidative modification can also be triggered by exposure of vascular cells to transition metals, which results in the oxidation modification of cholesterol, HDL and LDL. The concept that transition metals can trigger oxidation of LDL has been reviewed in the U.S. patent application Ser. No. 11/230,277 by Guilford, entitled “Combination and method using EDTA combined with glutathione in the reduced state encapsulated in a liposome to facilitate the method of delivery of the combination as an oral, topical, intra-oral or transmucosal for anti-thrombin effect and for anti-platelet aggregation and measurement of efficacy”.
The tripeptide L-glutathione (GSH) (gamma-glutamyl-cysteinyl-glycine) is well known in biological and medical studies to serve several essential functions in the cells of higher organisms such as mammals. It is functional when it appears in the biochemical form known as the reduced state (GSH). When oxidized, it forms into a form known as a dimer (GSSG).
Glutathione in the reduced state (GSH) in this invention functions as the specific substrate for the enzyme glutathione peroxidase, which enzyme functions to inhibit cholesterol, particularly HDL and LDL oxidation. The enzyme cooperates with the GSH to maintain the HDL and LDL in a reduced and functional state. Antioxidants such as Vitamin C or vitamin E will not cause that functionality. GSH also functions as an antioxidant, protecting cells against free-radical mediated damage, acting as a detoxifying agent by transporting toxins out of cells and out of the liver, and acting as or facilitating cell signals, particularly in the immune system. GSH is particularly active with respect to 2OH− radicals, because 2 GSH− each easily substitute their S—H bonds. Upon such substitution, the two “GS-molecules” bond to each other into an oxidized state forming GSSG, and the 2OH− radicals are reduced in charge and in number of free electrons by each hydroxyl radical bonding with an H atom from the thiol group of GSH. Most glutathione taken orally and even NAC taken orally are not presented in an intracellular context as reduced glutathione. There was and is a need for reduced glutathione and a means of transport of reduced glutathione. Further, the substance to address HDL and LDL oxidation must be able to interact with glutathione peroxidase that is on the surface of the cell.
A deficiency of glutathione (reduced) may lead to damage to cells and tissues through several mechanisms including the accumulation of an excess of free radicals which causes disruption of molecules, especially lipids causing lipid peroxidation, and which, combined with toxin accumulation, will lead to cell death. As a general term, these mechanisms are often referred to as oxidation or peroxidation. The lack of sufficient glutathione in the reduced state relative to the oxidized state may be due to lack of production of glutathione (reduced) or an excess of the materials such as toxins that consume glutathione (reduced). The lack of glutathione (reduced) may manifest as a systemic deficiency or locally in specific cells undergoing oxidation stress.
Hastings et al, U.S. Pat. No. 6,368,617 reference the combination of glutathione and CoQ10 in combination with 7-keto dehydroepiandosterone (7-keto DHEA) for a health promoting combination. There is no reference for the use of this combining of liposomal encapsulation of reduced glutathione combined with CoQ10 for prevention or treatment of oxidized cholesterol and its vascular effects.
Richardson et al U.S. Pat. No. 6,207,190 reference the use of an oral combination of at least 4 materials including folic acid, magnesium, lipoic acid and N-acetyl-cysteine which is used as a source of glutathione for the treatment of glaucoma, a disease of the eye. While their patent references the use of glutathione to stabilize Nitric Oxide (NO), there is no reference to its use systemically to limit damage to endothelial lining cells of arteries or prevent the oxidation of cholesterol, HDL and LDL or the deleterious sequence of events that occur related to these oxidation products, nor any reference to reduced glutathione.
A review of the literature reveals one study of the effect of the intravenous infusion of reduced l-glutathione (Intravenous refers to the method of administration of aqueous materials using an infusing tube inserted through a vein to facilitate direct venous system administration.) The study using the intravenous infusion of reduced glutathione given in a dose of 600 mg twice a day for a week suggested that there may be clinical benefit to the infusion of glutathione (Arosio). There is no reference to the use of an orally absorbable form of glutathione in the article and no reference to the use of liposomal glutathione. The article also has no reference to the reduction of oxidized lipids.
Rodriguez et al, in U.S. Pat. No. 6,773,719 reference the use of plain liposomes in the management of atherosclerosis. The mechanism involves the removal of the excess lipids by the liposomes. There is no reference to using reduced glutathione in the liposomes, and no reference as to how to enable liposomal reduced glutathione to cooperate effectively with GpOx. The mechanism of this invention is not direct to removal of excess lipids.
Williams, U.S. Pat. No. 6,367,479 references the use of large unilaminar liposome that is single walled, vesicles like liposomes for the transport of cholesterol from arterial vessels back to the liver. However, there is no reference to liposomes containing reduced glutathione or to the use of liposomes containing reduced glutathione for the management of disease related to oxidation of lipids such as cholesterol, HDL or LDL.
Demopoulos et al in U.S. Pat. No. 6,350,467 references the use of a pharmaceutical preparation of a combination that includes glutathione in a powdered form for the treatment of atherosclerosis. However there is no reference for the use of the preparation for management of oxidized lipids such as cholesterol, HDL or LDL. There is also no mention of the use of the preparation in a liposome nor the use of reduced glutathione in a liposome.
Smith, U.S. Pat. No. 6,764,693 references the use of liposomes containing a combination of glutathione in combination with at least one other antioxidant material to increase intracellular and extra cellular antioxidants. There is no reference to liposomes containing reduced glutathione which is what is needed to cooperate with glutathione peroxidase in order to prevent oxidation of HDL and LDL, nor is there any reference to the use of liposomes containing reduced glutathione for the management of disease related to oxidation of lipids such as cholesterol, HDL or LDL.
Meyerhoff, U.S. Pat. No. 6,469,049, Oct. 22, 2002, references the combination of lipoic acid and glutathione or CoQ10 for the management of central nervous system injuries, but does not reference the use of glutathione and CoQ10 together nor in the management of vascular disease. Likewise, there is no reference to the combination of glutathione, CoQ10 and statins, nor reduced glutathione, CoQ10 and statins, nor liposomal encapsulation of reduced glutathione, CoQ10 and statin for the management of atherosclerosis or elevated cholesterol or oxidized cholesterol or oxidized LDL or HDL.
Cooke in US patent application 20020151592 references the use of arginine or lysine to increase nitric oxide production for vasodilation purposes and he also references the use of glutathione as an antioxidant used in conjunction with arginine. However, there is no mention of the use of reduced glutathione, nor is there mention of the use of liposomal encapsulation of glutathione or reduced glutathione to supply the reduced glutathione needed for maintaining adequate GSNO formation. The current invention is also distinguished from the reference by Cooke in that the current invention supplies reduced glutathione in a form that is readily absorbable into the body, where plain powdered glutathione is not well absorbed as reviewed in the previously referenced application, Guilford U.S. application Ser. No. 11/163,979 filed Nov. 6, 2005.
Oxidative stress occurs when there is an imbalance between free radical production and antioxidant capacity (Penckofer). This may be due to increased free radical formation in the body and/or loss of normal antioxidant defenses. Oxidative stress is defined as excessive production of reactive oxygen species (ROS) in the presence of diminished antioxidant substances (Opara).
Reactive oxygen species (ROS) are generated as by-products of normal cellular metabolism, primarily in the mitochondria (Miyamoto), or as a result of external biochemical stress or in response to inflammatory stimuli. When the cellular production of ROS exceeds the cell's antioxidant capacity, cellular macromolecules such as lipids, proteins and DNA can be damaged. Damage to these biochemicals leads to the concept of ‘oxidative stress’, which is thought to contribute to aging and pathogenesis of a variety of human diseases. The body's defense against oxidative stress is accomplished by interconnecting systems of antioxidant micronutrients (vitamins and minerals) and enzymes. While the vitamins act as donors and acceptors of ROS, minerals regulate activity of the enzymes (Opara).
A combination of factors cause atherosclerosis. The imbalance between circulation levels of cholesterol transported in HDL versus LDS is intimately associated with dysfunction in the lining of arteries and oxidation stress in the arterial wall cells. This dysfunction is closely related to inflammation. Dyslipidemia, oxidation stress and inflammation are closely interrelated to the development of atherosclerosis. OxLDL leads to a series of inflammatory events related to immune modulator release from macrophages that ingest the oxidized lipid. It has been demonstrated that most of the proinflammatory properties of oxLDL result from products of the oxidation of LDL. This perspective gives the view that atherosclerosis is a chronic inflammatory disease of the arterial wall mediated by oxLDL in concert with a range of proinflammatory agents (Kontush). Based on the unpublished study by Aviram, discussed herein, called “The effect of liposomal glutathione on the oxidation of the cholesterol components known as Low density lipoprotein (LDL) and high density lipoprotein (HDL),” lack of available reduced glutathione to maintain normal antioxidant function in LDL and lack of reduced glutathione to maintain the normal antioxidant function of HDL appears to be intimately related to maintenance of normal artery function and development of atherosclerosis. The absence of adequate glutathione is the common factor resulting from dyslipidemia, and leads to oxidation stress. Thus adequate biochemically reduced glutathione mediates the inflammation associated with atherosclerosis.
The present invention, liposomal reduced glutathione, offers a novel method to maintain cholesterol, LDL and HDL in their reduced, functional state, which will delay or avoid the progression of events leading to atherosclerosis. The current invention offers a method of oral administration of an absorbable material that is needed to supply the physiologic substrate needed to work with glutathione peroxidase in the maintenance of normal artery lining cell function and potentially slow, stop or reverse vascular disease by deterring creation of Oxidized LDL or HDL.
Oxidation stress is related to many disease states. Depending on the gradation of the stress effect a diverse array of disease states are related to oxidation stress. These diseases include cardiovascular diseases, neurological diseases, malignancies, renal diseases, diabetes, inflammatory problems, skin diseases, aging, respiratory diseases, liver diseases and different types of viral infections (Irshad).
Normal metabolism forms free radicals, leading to oxidation. These metabolites either from oxygen or nitrogen are called pro-oxidants. The stress component refers to changes that put higher demands on the available antioxidants. The demand for increased antioxidants may pull energy away from the production of other cell metabolites and/or slow their formation. When the state of oxidation increases it can begin to interfere with cell function. With oxidation stress the changes could still be reversible, even if only reversible in part, with the addition of the appropriate antioxidant.
As the cell changes and oxidation damage occurs, the change and damage become less reversible and begin to increase to include macromolecules in the cells including protein, DNA and the lipids found in membranes. The result is initially seen as cell damage and when widespread can be seen as tissue damage.
To counterbalance the deleterious effects of oxidation stress, the body uses an array of nutrients and metabolites called antioxidants. Antioxidants are provided by the normal metabolism of the body or from outside sources such as vitamin C. Antioxidants include superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, as well as minerals like Se, Mn, Cu and Zn, and vitamins like vitamin A, C and E. Other compounds with antioxidant activity include glutathione, flavonoids, bilirubin and uric acid (Irshad). Selenium is contemplated as part of the preferred composition of the invention as selenium has been shown to increase the availability of glutathione peroxidase in the serum of individuals under oxidative stress and who had been observed to have low glutathione system function (Hussein).
There is a growing awareness that the glutathione related antioxidant system plays a key role in the prevention of oxidation stress. For example, in patients with coronary artery disease, a below-normal level of the glutathione related enzyme called glutathione peroxidase-1 has been found to be independently associated with an increase risk of cardiovascular events (Blankenberg). Glutathione plays such a crucial role in management of oxidation stress that deficiency of glutathione is now being referred to as part of the definition of oxidation stress. Some articles use the general definition that describes the situation in the following manner, “Oxidative stress has been defined as a loss of counterbalance between free radical or reactive oxygen species (ROS) production and antioxidant systems.” (Dursun). More recently, many authors are defining oxidation stress as dependent on the availability and function of the glutathione system, as exemplified by the statement from a recent article, “Oxidative stress may be viewed as an imbalance between reactive oxygen species (ROS) and oxidant production and the state of glutathione redox buffer and antioxidant defense system” (Tappia). Another example is the description of oxidation stress found in diabetes, “Patients affected by diabetes mellitus have oxidative stress with an impaired glutathione (GSH) redox state.” (Bravi)
In spite of the awareness of the role of oxidation stress in disease there is a persisting controversy in regard to the role of oxidation stress in the development of atherosclerosis. While there is adequate research demonstrating the role of oxidation stress related to atherosclerosis, antioxidants such as vitamin E or beta-carotene (Clarke) or vitamins A, C, and E, and beta carotene (Hasnain) have not been effective in and of themselves in reducing cardiovascular death and morbidity in human clinical trials. This has led many investigators to question the importance of oxidation stress in human atherosclerosis (Madamanchi). It is proposed in this application that the failure to observe benefit of antioxidant therapy in atherosclerosis may be due to the lack of the appropriate antioxidant for the situation.
Much of the confusion regarding the role of glutathione in management of vascular disease may be related to two concepts. The first is that it has been previously thought that glutathione peroxidase is not found outside the cell or in extracellular fluids (Stocker). The second concept is that human clinical studies with antioxidants in vascular disease in general have been disappointing (Cynshi). However, there have been no prior studies of the use of an oral preparation of glutathione in its reduced form or the use of liposomal encapsulation of reduced glutathione for the prevention of atherosclerosis.
A human study observing the relationship between oxidation status and thickening of the lining of the carotid artery, measured by ultrasound has established a relationship with the level of available reduced glutathione (Ashfaq). Carotid artery intimal thickening has been shown to correlate with both risk factors of atherosclerosis and the presence of coronary artery atherosclerosis (Crouse). The factors correlated with increased carotid artery intimal thickening were the absolute level of reduced glutathione and the ratio of reduced glutathione (gsh) and oxidized glutathione (gssg). In the Ashfaq study, the single variable that most closely predicted increased intimal thickening was the ratio of reduced glutathione to oxidized glutathione or gsh/gssg. The more reduced glutathione relative to oxidized glutathione that was available, the less intimal thickening was observed.
Low Density Lipoproteins commonly known as LDL refers to a class of lipoprotein particles. The lipid component is composed of the fatty acid molecules associated with cholesterol. The protein component consists of apoprotein B-100 and Apo E. LDL particles are identified by their size (18-25 nm diameter) and their components. Apolipoproteins of the B category are found in LDL and Apoprotein B-100 is the key protein associated with LDL. This protein is retained within the artery wall in close association with the proteoglycans found in the artery wall and play an important role in the formation of atherosclerosis. Therefore, the term LDL will refer to “native” apolipoprotein B-100, and oxLDL will refer to oxidized apoprotein B-100. Further, these terms will refer to the additional proteoglycans associated with the sites of atherosclerotic lesions and lipoprotein deposition.
High Density Lipoprotein (HDL) is a smaller molecule (8-11 nm diameter). These particles function to carry cholesterol from tissues that do not need cholesterol back to the liver. This is a key function of HDL related to the preventing the accumulation of too much LDL in the cells lining arteries. As the HDL particles have the potential to remove cholesterol from plaque in the arteries it is also known as “good cholesterol”. The density of HDL derives from its small size and the high proportion of protein that they contain. These lipoproteins play a role in gathering the cholesterol. For example, HDL contains an apolipoprotein (apo) called A-I. ApoA-I is the major protein component of antiatherogenic high-density lipoprotein (HDL). As the HDL circulates it may increase in size as it incorporates more molecules of cholesterol into its structure. One of the methods of antioxidant function of HDL is through its ability to bind transition metal ions such as iron and copper, which are potent catalyzers of LDL oxidation (Kunitake). A low level of HDL is statistically correlated as a risk factor for coronary artery disease (Kontush). Even in individuals on cholesterol lowering therapy, the level of HDL remains a significant predictor of cardiovascular events. Thus, the ability of HDL to function in its native or biochemically reduced state is critical to the normal function and the avoidance of atherosclerosis. Oxidation of HDL impairs the ability of HDL to promote cholesterol efflux by the ATP-binding cassette transporter A-1 pathway (Navab). HDL can become oxidized through oxidation stress, diabetes and exposure to free radicals generated from transition metals and oxHDL does not function as efficiently as the native HDL. Thus, there is increasing attention focused not only on the quantity of HDL, but also the oxidation state of HDL.
The term oxidation stress is used to define the metabolic events associated with damage from free radicals related to stress in cells and organ tissues. The concepts behind thus descriptive are familiar to persons reasonably skilled in the art; yet defining these events remains difficult. No literature or prior art has suggested how to manage that oxidation state of HDL nor has any prior art or literature suggested using glutathione reduced or an oral liposomal encapsulation of reduced glutathione to alter the oxidation state of HDL.
HDL has become recognized as playing a key role in both removing excess LDL and maintaining LDL in the non-oxidized state. Thus, methods that maintain normal function of HDL play a vital role in maintaining both normal LDL levels, as well as preventing the oxidation of LDL. The combined effect of the present invention liposomal glutathione in maintaining normal reductive efficacy of glutathione peroxidase (GPx) associated with cholesterol, LDL and HDL as well as its action on Phospholipid Hydroperoxide Glutathione Peroxidase (PH-GPx), an antioxidant enzyme that is able to directly reduce lipid peroxides even when they are bound to cellular membranes, creates the novel and surprising effect of decreasing lipid peroxidation by supplying the natural component, reduced glutathione, which the stress of excess toxins has diminished. Prior to the present invention, there has been no method of providing systemic availability of reduced glutathione to support these natural systems, and particularly to support both intracellular and cellular-membrane lipids and lipid derivatives. The surprising finding that glutathione peroxidase is present on LDL and HDL allows the present invention to provide a novel route for support cooperative effect that is normally present in the body to protect against damage from the formation of oxLDL. Thus, the present invention, liposomal glutathione (reduced) both prevents the oxidation of LDL, as well as maintaining HDL in a non-oxidized state so that its function is also maintained on both the cell membrane and in the cell. The present invention is a novel composition that is convenient, stable and orally available for maintaining normal levels of reduced glutathione even in conditions where the ability of the system to maintain natural glutathione levels has been overburdened or compromised. In situations such as an excess of oxidized LDL, excess cholesterol or other oxidizing stress situation, the current invention provides a convenient method of maintaining LDL in the non-oxidized state.
The use of a precursor of glutathione, N-acetyl-cysteine (NAC) has been referenced by Kindness, Guilford et al in US Patent application published as 20020182585, published Dec. 5, 2002, as a preferred mode of an invention using NAC to build glutathione. While it has been observed that cysteine is an amino acid component of the tripeptide glutathione, recent observations such as reviewed in Example 2, suggest that the direct delivery of reduced glutathione is more effective. It is known that cysteine can also be directed to form taurine. Taurine is one of the most abundant amino acids in the body and in certain situations the formation of glutathione can be altered in favor of the formation of taurine. Taurine does have antioxidant properties, but does not interact with the enzyme system that supports the glutathione antioxidant system, such as glutathione peroxidase. The preference for the production of taurine over glutathione is reported to occur with inflammation (Santangelo) and toxins such as alcohol (Jung) and in this application is reported to occur with in the presence of an excess burden of mercury in the human system. For the purpose of this discussion this switch in biochemical pathways will be referred to as the “taurine shunt”, and is illustrated in FIG. 5. The abbreviations in FIG. 5 are as follows: THF: tetrahydrofolate; MS: methionine synthase; BHMT: betaine-homocysteine methyltransferase; MAT: methionine adenosyltransferase; SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine; SAHH: SAH hydrolase; ADA: adenosine deaminase; AK: adenosine kinase; CBS: cystathionine beta synthase; B12: cyanocobalomine; meB12: methylcobalamine; 5-CH3 THF: 5-Methyltetrahydrofolate.
This finding of preference for the production of taurine over glutathione is significant as the shunt from glutathione production to taurine may occur locally in tissues and cells and may be a contributing factor to the local as well as systemic reduction of reduced glutathione found in atherosclerosis. In addition, an excess of taurine as evidenced by increased excretion of taurine in the urine has been observed by the applicant in children with autism, who are documented to have low levels of glutathione systemically.
The presence of the “taurine shunt” makes the liposomal encapsulation of reduced glutathione the preferred mode for the delivery and maintenance of reduced glutathione for the present invention.
The combination of an HMG-CoA reductase inhibitor and CoQ10 has been referenced by Brown in U.S. Pat. No. 4,933,165, Jun. 12, 1990. There is no reference to liposomes in Brown '165 nor to the use of reduced glutathione in combination with a statin, nor with a statin and CoQ10. The use of CoQ10 with a statin is distinctly different from the combination of liposomal glutathione and statin. The CoQ10 is added to replace the loss of CoQ10 that occurs with the use of the HMG-CoA reductase inhibitor. In the present invention embodiment, the unique concept is that the liposomal glutathione is added to enhance the effect of maintaining cholesterol and LDL in the biochemically reduced state. By maintaining the reduced state, that is avoiding the oxidation of Cholesterol or the oxidation of LDL, the most beneficial effect of reduced glutathione and a statin working together is achieved. Further, the goal of lessening the formation of atherosclerosis will be achieved using a lower dose of the statin drug and thereby minimizing the side effects of the statin. The current goal of treatment by a statin has been to lower the level of LDL to lessen the progression of atherosclerosis. The goal of the combination in the current invention is to lessen the progression of atherosclerosis by lessening the formation of oxLDL, and facilitating the function of HDL. As has been noted, decreased oxLDL is advantageous in avoiding atherosclerosis. There is no reference to the combination of HMG-CoA reductase inhibitor, CoQ10 and glutathione as related in the current invention.
The intravenous administration of reduced glutathione has been reported to have benefit in improving blood flow in peripheral vascular disease (Arosio), however there has been no reference to the mechanism of maintaining or returning lipid peroxidation to the reduced (non-oxidized) state using glutathione as a substrate for the glutathione peroxidase that has been found to be associated with native cholesterol, LDL and HDL as reported in this invention. The interaction of glutathione naturally with glutathione peroxidase associated with these lipids in the circulation represents a critical step in the prevention of atherosclerosis. The novel and surprising aspect of being able to supply reduced glutathione in an orally absorbable liposome creates an exciting new method for the study and management of atherosclerotic vascular disease that has not been previously reported. The ability to supply reduced glutathione orally in the present invention represents a novel method of management of oxidation of lipids such as cholesterol, LDL and HDL and represents a significant advantage in disease management.
There is evolving in the literature the knowledge that a significant number of cardiovascular events occur in individuals with normal levels of HDL and LDL (Navab). This observation has stimulated investigations for biomarkers with higher predictive value. Recent articles have proposed that the level of oxidized LDL has high correlation with progression of vascular disease. There have been no reports of plain glutathione being capable of absorption in the human to provide adequate levels of support for the glutathione system. The liposomal encapsulation of reduced glutathione provides a novel method for achieving effective systemic and intracellular delivery of glutathione (reduced).
The function of LDL in the system is to carry cholesterol and triglycerides away from cells and tissue that produce more than they use, such as the liver, to cells and tissues that take up cholesterol, such as arteries. The normal state of these lipids is the biochemically reduced form. When altered by oxidation, these lipoproteins are identified as oxidized LDL or oxLDL.
LDL is often referred to as “bad cholesterol” as it has a statistical association with cardiovascular disease. The inventors believe it is not the LDL that is bad, but where and what state of oxidation the LDL molecule is in that may be the more compromising component of LDL metabolism. In terms of triggering the artery wall changes found in atherosclerosis, oxidized LDL is becoming known as the trigger for atherosclerosis. OxLDL has been shown to be a strong and independent risk factor of vascular disease such as coronary heart disease even in apparently healthy individuals (Meisinger). Thus, the search for a trigger for developing atherosclerosis, is shifting focus from the management of the LDL to the question of how to manage the oxidation state of LDL. No literature or prior art has suggested how to directly manage the oxidation state of LDL nor has any prior art or literature suggested using glutathione reduced or an oral liposomal encapsulation of reduced glutathione to alter the oxidation state of LDL.
Reactive Oxygen Species (“ROS”) exert some functions necessary for cell homeostasis maintenance, but loss of the balance between the anti- and pro-oxidant states results in pathology. ROS mediated lipid peroxides are of critical importance because they participate in chain reactions that amplify damage to biomolecules including membranes and DNA. DNA attack gives rise to mutations that may involve tumor suppressor genes or oncogenes, and this is an oncogenic mechanism (Cejas). OxLDL has been observed to increase cell proliferation (Zettler) and has been considered one stimulant for the proliferation of smooth muscle. While it has been observed that oxLDL induces an increased expression of both the promoters and inhibitors of the cell cycle. Activating inducers and inhibitors should allow for self regulation of cell growth. The generalized induction of both cell cycle inducers and inhibitors with cooperation among the cell cycle regulators is consistent with the slower, non-malignant cell growth typical of an atherosclerotic plaque. While balance between stimulation and inhibition occurs in other non-malignant situations such as the rapid cell proliferation seen in liver regeneration, malignant cell growth is typically characterized by high levels of the one or more of the cell cycle inducers and low or absent cell cycle inhibitors.
Contrary to the unregulated cell cycle proliferation in cancer, one difference between regulated cell cycle proliferation in normal or atherosclerotic cells and unregulated cell cycle proliferation in cancer cells and in atherosclerotic cells is that the there is both an increase in expression and nuclear translocation of both the activators and inhibitors of the cell cycle. It is conceivable that the continued exposure of cell nuclei to excessive oxLDL could result in abnormal modulation of cell cycle reproduction, which in cancer means little or no modulation of reproduction.
Oxidation stress indicators have been found in various cancer cells and it is postulated that the redox imbalance may be related to stimulation of changes not only in the affected cells, but also in the body response toward cancer. Oxidative DNA abnormalities are noted in many tumors and it appears that oxidative DNA damage is linked with the process of initiation of cancer. As a variety of transition metals such as iron, copper, cadmium, arsenic and nickel are involved in the formation of the free radicals via reactions such as the Fenton reaction, a deficiency of reduced glutathione and the corresponding ability to modulate free radical formation may play a critical role in the induction of cancers. Thus, methods that maintain normal levels of metals such as iron and copper and remove the abnormal metals will be of significant benefit in avoiding the oxidative stimulation that can lead not only to atherosclerosis, but also to cancers. The use of liposomal glutathione and EDTA has been previously reviewed by Guilford, U.S. patent application Ser. No. 11/230,277 filed Sep. 20, 2005.
Additional impact from metals is observed with metals such as mercury, cadmium, nickel and arsenic, each of which also depletes glutathione (Valko, 2005). It is probable that metal induced oxidation stress may create an oxidative environment favorable to the development of cancer (Valko, 2006).
Patients with cancer have an increased amount of oxidation stress related to the disease process. The addition of chemotherapy agents increases the oxidation stress and risk of side effects such as neuropathy. For example, the occurrence of neuropathy has been found to limit the number of treatments with cisplatin. The use of intravenous reduced glutathione concurrent with the administration of cisplatin has been observed to allow increased dose intensity of treatments and to not interfere with the efficacy of the drug (Di Re). No prior art suggests the novel use proposed herein of the use of an oral liposomal preparation of reduced glutathione as described in this application for the reduction of side effects during chemotherapy. In addition, there are no reports of orally effective liposomal glutathione being used post chemotherapy to help restore normal glutathione levels.
Example 3 illustrates the effect that extensive disease related to cancer can have on the patient and the impact that the current invention may have on the treatment of an individual with cancer that can occur by the administration of the invention, liposomal glutathione. Cancer has been shown to be associated with an increase in oxidation stress in general particularly oxidized LDL. It is proposed that the current invention be considered as a component of cancer therapy, especially after therapies or at a stage of progression in which oxidation stress may have become overwhelming. While the dramatic improvement experienced by the individual was not permanent, it does demonstrate that the use of the current invention has relevance to the clinical management of cancer.
Oxidation stress creates a series of concurrent events, which affect the status of the cells lining arteries. While the oxidation stress effects on lipids are occurring another effect is the decrease in availability of nitric oxide produced by the endothelial cells.
Nitric oxide has the capacity to cause vasodilation, but if not stabilized, can also contribute the formation of free radicals called Reactive nitrogen species, which will actually contribute further to the cascade of oxidation locally in an artery. Nitric oxide has been shown to react with glutathione, which creates S-nitrosoglutathione (GSNO). The GSNO molecule is more stable than NO and has been demonstrated dilating effects directly in the lung, where it has been shown to dilator activity (Que). This mechanism is reviewed in Guilford, U.S. Application 60/596,171 filed 6 Sep. 2005 entitled “Method for the Treatment of Infection with HHV-6 Virus and the Amelioration of Symptoms Related to Virus Using Liposomal Encapsulation for Delivery of Reduced Glutathione and PCT/US2006/34648 filed 6 Sep. 2006.
In artery disease the presence of oxidized LDL creates an increased demand on the availability of reduced glutathione, leaving less glutathione available to create the GSNO molecule needed to stabilize nitric oxide (NO). Normally, the expansion of the artery prevents the increase in pressure that accompanies the contraction of the heart muscle pumping blood through the artery system. Without this vasodilatation capacity, an increase in the pressure in the artery will occur as the artery acts like a solid pipe. This increased pressure within the pipe-like artery is what we call hypertension. The lack of vasodilating action by GSNO leads to decreased relaxation in the artery and manifests as increased blood pressure, known has hypertension. An additional mode of action of the present invention is the introduction of reduced glutathione via the liposomes to provide the glutathione necessary for the creation of GSNO at sites of nitric oxide production and glutathione deficiency. This will be particularly beneficial in individuals with either elevations of oxidized LDL or increased LDL, which leads to increased oxLDL.
The ability to increase the efficacy of the natural mechanism of the blood pressure maintenance or to naturally facilitate the function of medications or supplements designed to maintain normal blood pressure is a novel and unexpected finding related to the use of liposomal glutathione. As reviewed in examples 4 and 5, the liposomal glutathione is effective in combination with either a nutrient that increases nitric oxide such as arginine or lysine or in combination with a prescription medication such as lisinopril in combination with arginine and the liposomal glutathione.
The preferred dose of the combination of the invention, liposomal glutathione, and arginine is liposomal glutathione 800 mg in combination with each 450 mg capsule of arginine. This combination may be taken once or twice a day as needed to maintain a normal blood pressure.
The preferred combination of the invention in combination with blood pressure medication is with lisinopril 20 mg daily, in combination with 800 mg of liposomal glutathione. This may be combined with arginine 450 mg as needed.
Additionally, in the situation where there is oxidized LDL being formed in the system an excess of reduced glutathione may need to be supplied in addition to the nitric oxide enhancing agent to maintain adequate formation of GSNO. The formation of GSNO is the normal reaction that occurs in the body to maintain the vasodilating properties of NO and requires a continuous supply of reduced glutathione to be formed. An excess of oxidation stress will create the situation where the body needs to supply an excess of reduced glutathione via the current invention, liposomal glutathione, to be able to utilize the nitric oxide that is formed.
The management of elevated blood pressure uses medications that fall into the following categories:
1. Diuretics                chlorthalidone (Hygroton), furosemide (Lasix), hydrochlorothiazide (Esidrix, Hydrodiuril, Microzide), indapamide (Lozol), metolazone (Mykrox, Zaroxolyn)        
2. Potassium-sparing diuretics                amiloride hydrochloride (Midamar), spironolactone (Aldactone), triamterene (Dyrenium)        
3. Combination diuretics                amiloride hydrochloride+hydrochlorothiazide (Moduretic), spironolactone+hydrochlorothiazide (Aldactazide), triamterene+hydrochlorothiazide (Dyazide, Maxzide)        
4. Beta-blockers                acebutolol (Sectral), atenolol (Tenormin), betaxolol (Kerlone), bisoprolol fumarate (Zebeta), carteolol hydrochloride (Cartrol), metoprolol tartrate (Lopressor), metoprolol succinate (Toprol-XL), nadolol (Corgard), penbutolol sulfate (Levatol), pindolol (Visken), propranolol hydrochloride (Inderal), timolol maleate (Blocadren).        
5. Angiotensin Converting Enzyme Inhibitors (ACE inhibitors)                benazepril hydrochloride (Lotensin), captopril (Capoten), enalapril maleate (Vasotec), fosinopril sodium (Monopril), lisinopril (Prinivel, Zestril), moexipril (Univasc) quinapril hydrochloride (Accupril), ramipril (Altace), trandolapril Mavik.        
6. Angiotensin II receptor blockers                candesartan (Atacand), irbesarten (Avapro), losartin potassium (Cozaar), valsartan Diovan        
7. Calcium channel blockers                amlodipine besylate (Norvasc), diltiazem hydrochloride (Cardizem CD, Cardizem SR, Dilacor XR, Tiazac), felodipine (Plendil), isradipine (DynaCirc, DynaCirc CR), nicardipine (Cardene SR), nifedipine (Adalat CC, Procardia XL), nisoldipine (Sular), verapamil hydrochloride (Calan SR, Covera HS, Isoptin SR, Verelan).        
8. Alpha blockers                doxazosin mesylate (Cardura), prazosin hydrochloride (Minipress), terazosin hydrochloride (Hytrin).        
9. Combined alpha and beta-blockers                carvedilol (Coreg), labetolol hydrochloride (Normodyne, Trandate).        
10. Central agonists                alpha methyldopa (Aldomet), clonidine hydrochloride (Catapres), guanabenz acetate (Wytensin), guanfacine hydrochloride (Tenex).        
11. Peripheral adrenergic inhibitors                guanadrel (Hylorel) guanethidine monosulfate (Ismelin), reserpine (Serpasil).        
12. Blood vessel dilators                hydralazine hydrocholoride (Apresoline), minoxidil (Loniten).        
The preferred mode of the invention is the combination of lisinopril 20 mg and Liposomal glutathione 800 mg (2 teaspoons). Additional preferences include the other agents in the Angiotensin Converting Enzyme (ACE) Inhibitor category of drugs.
The invention, liposomal glutathione, is also proposed in combination with the nitric oxide enhancing agents such as the nutrients 1-arginine and 1-lysine in order to facilitate the formation of GSNO, which is needed for vasodilation.
It is proposed that the invention, liposomal glutathione, in combination with an antihypertensive agent such as lisinopril and arginine to allow a more efficient blood pressure lowering using a lower dose of the antihypertensive agent as illustrated in example 5.
The liposomes and the encapsulation of reduced glutathione used in the current invention are described in Guilford, U.S. patent application Ser. No. 11/163,979 filed Nov. 6, 2005, published May 11, 2006 as 20060099244 which is incorporated in its entirety. While the preferred embodiment of the invention is the preferred embodiment listed in Guilford, U.S. patent application Ser. No. 11/163,979, the other methods of liposomal encapsulation of the invention as described in Guilford, U.S. patent application Ser. No. 11/163,979 as well as in Keller et al, U.S. Pat. No. 5,891,465, Apr. 6, 1999, Keller et al U.S. Pat. No. 6,958,160, Oct. 25, 2005, and Keller US Patent application 20020039595, published Apr. 4, 2002 are incorporated into this description.
The current application is prompted after reviewing the unpublished findings of a study on the activity of a liposomal encapsulation of reduced glutathione on the stabilization of cholesterol, LDL and HDL when they were exposed to agents known to create rapid oxidation of these materials. As will be reviewed, the oxidation of Low Density Lipoprotein (LDL) with the oxidized LDL referred to as oxLDL, is significantly slowed by the addition of liposomal reduced glutathione, the current invention, which utilizes glutathione peroxidase as a key to enable reduced glutathione to function to prevent oxidation of cholesterol. Exporting reduced glutathione effectively into the blood system and into cells has not been previously feasible except by intravenous means. By liposomal encapsulation, this invention enables that export of reduced glutathione into the blood system making it available to cells d and cell membranes. The liposomal encapsulation also allows the availability of that reduced glutathione to cooperate with Glutathione Peroxidase to impeded undesired oxidation of LDL and HDL cholesterol. In the presence of high fat diet or high lipid content of blood, the normal glutathione formation capacity of the system is often exceeded requiring an outside source of intracellular glutathione to maintain the antioxidant status of the system. This invention provides a novel and surprising answer to prevent or slow the oxidation of LDL, which is associated with atherosclerosis.
As related in the unpublished Aviram study, the novel finding of glutathione peroxidase related to native cholesterol, HDL and LDL suggests that the availability of reduced glutathione is critical for maintaining the normal state of function of these materials. Other antioxidants may provide indirect support for this function, but reduced glutathione functions much more efficiently.